Melanoma is one of the most rapidly growing cancers worldwide with an estimated 8,420 deaths and 62,480 new cases in the United States in 2008 alone. While melanoma can be surgically removed when detected early, there is no cure for advanced or metastatic disease. Currently offered therapies such as decarbazine or IL-2 demonstrate response rates of only 15 to 25%. Hence, effective treatment of this malignancy represents a serious unmet medical need. Melanoma has been considered to be an appropriate target for immunotherapies due to the observed immune infiltrate at the primary tumor that has been associated with spontaneous regressions of distant metastasis. It has also been demonstrated that melanoma can spontaneously induce melanoma-specific cellular and humoral immunity. In fact, evidence for clinical benefit of a therapeutic melanoma vaccine was recently demonstrated in a prospective, randomized Phase 3 clinical trial, further suggesting a critical role of the immune system in control of this disease. Recent advances in the field of bacterial-based vaccine vectors have shown promising results for cancer-targeted immunotherapy. One such example is the use of live-attenuated and killed strains of the intracellular bacterium Listeria monocytogenes (Lm). Lm represents an attractive platform for therapeutic melanoma vaccine due to its ability to induce robust innate immunity as well as acquired CD4+ and CD8+ T-cell immunity. Lm is a pathogenic infectious agent in man, and in order to provide an appropriate therapeutic window, we have developed two highly attenuated vaccine platforms. The first is live-attenuated Lm in which two virulence genes have been completely deleted from the bacterial chromosome (Lm ?actA?inlB). The second platform is based on Lm that is inactivated in such a way to prevent its replication while retaining its metabolic activity. These "killed but metabolically active" (KBMA) Lm are still able to induce robust cellular immunity and therefore provide a promising approach for therapeutic cancer vaccine development. The live-attenuated Lm strain, on which the KBMA Lm is based, formed the foundation for three Phase 1 clinical trials where 30 patients were dosed safely with up to 1 x 109 live-attenuated bacteria for the treatment of advanced cancers and chronic hepatitis C infections. The overall goal of this proposal is to develop and select a clinical vaccine candidate strain based on photochemically-inactivated KBMA Lm that express the cancer-testis antigens NY-ESO-1 and MAGE-A3 as a therapeutic melanoma vaccine and ultimately evaluate the safety and efficacy in human clinical trials. To this end, we propose the following aims: Aim 1. Construct and select a bivalent clinical vaccine candidate strain based on KBMA Lm that encodes NY-ESO-1 and MAGE-A3 (KBMA Lm MAA);Aim 2. Construct and characterize modified KBMA Lm MAA to enhance their ability to stimulate dendritic cells (DCs);and Aim 3. Select a single clinical vaccine candidate strain as in vivo DC activation and antigen delivery vehicle based on in vitro and in vivo immunogenicity for further development. PUBLIC HEALTH RELEVANCE: The incidence of melanoma is increasing at an alarming rate and yet for advanced or metastatic disease no effective treatments are available. It has been appreciated that the patient's own immune system plays a role in the control of this disease. In fact, spontaneous regressions are more frequently reported in melanoma compared to other cancers and these regressions correlate with an immune infiltrate at the site of the primary tumor. Recent advances in immunotherapy and encouraging results from a Phase 3 clinical trial demonstrating increased progression free survival suggest that therapeutic vaccines which induce multi- functional T cell immunity may be effective for the treatment of advanced or metastatic melanoma. The overall goal of this proposal is to develop a therapeutic vaccine for melanoma that is based on inactivated strains of recombinant Listeria monocytogenes expressing melanoma-associated antigens.